There are a multitude of schemes that have been reported and are currently used for the cloning of DNA. (See for example, Molecular Cloning A Laboratory Manual by T. Maniatis, E. Fritsch and J. Sambrook, Cold Spring Harbor Laboratory Publications, Cold Spring Harbour Laboratory, Box 100, Cold Spring Harbor, N.Y.) Most of these processes depend on techniques that do not select for a specific DNA fragment unless that fragment happens to already have a selectable marker. Thus, once a mixture of restriction fragments are ligated to a specific vector and transformed into a recipient bacterium, hundreds and perhaps thousands of transformants must then be screened to identify the clone of interest. This random, or shot gun approach as it is referred to, is very time consuming. The only other way to clone a specific fragment of DNA that does not have a selectable genetic marker on it requires that the fragment must be relatively pure prior to cloning. However, it would also be a very time consuming process to identify and purify the specific fragment. A further disadvantage of current cloning techniques becomes evident when DNA from a Gram positive bacterium, such as Bacillus subtilis is being cloned into E. coli, a Gram negative bacterium. In this case, there is the possibility that the desired genetic information encoded in the cloned fragment will not be expressed and thus can only be screened for by hybridization to specific probes. If expression does occur, but involves proteins that are normally excreted or located on the cell surface of the Gram positive bacterium, subsequent passage through the Gram negative cell envelope may not be possible.
There are four types of vector systems generally used to clone fragments of DNA into E. coli. They are plasmids, bacteriophage .lambda., cosmids and bacteriophage M13. Each vector system has particular features which make them useful for different purposes. They also share several common features. They can replicate autonomously in E. coli, they can be easily separated from bacterial nucleic acids and purified, and they contain regions of DNA that are not essential for propagation and into which foreign DNA can be inserted.
Cloning in plasmid vectors exemplified by Cohen/Boyer in U.S. Pat. No. 4,237,224, is in principle strightforward. The plasmid DNA is cleaved with a restriction endonculease and joined in vitro to foreign DNA. The recombinant plasmids that result, are then used to transform bacteria. In practice the plasmid vector must be chosen carefully for the particular cloning experiment in order to minimize the effort necessary to identify and characterize the DNA fragment of interest. The major difficulty is to distinguish between plasmids that contain the DNA fragment of interest from those that contain other pieces of foreign DNA and plasmid vectors that have recircularized.
The use of bacteriophage .lambda. as a cloning vector was first demonstrated by N. E. Murray and K. Murray, (see Nature, 251: 476, 1974) and A. Rambach and P. Tiollais (see Proc. Nat. Acad. Sci., 71: 3927, 1974) Cloning with bacteriophage involves several steps. The bacteriophage vector DNA is digested with the appropriate restriction enzyme and ligated to fragments of foreign DNA having compatible termini. The resulting recombinant DNA's are packaged in vitro into viable bacteriophage particles that form plaques on the appropriate hosts. Recombinant phages carrying the desired foreign DNA are identified by procedures involving nucleic acid hybridization. There is no single bacteriophage .lambda. vector suitable for cloning all DNA fragments. It is therefore necessary to choose carefully among the various bacteriophage vectors for the one best suited.
Cosmids were first developed by Collins and Hohn (see Proc. Nat. Acad. Sci., 75: 4242, 1978) and are vectors specifically designed for cloning large fragments of eukaryotic DNA. The essential components of cosmids are a drug resistance marker, plasmid origin of replication, one or more restriction sites for cloning, a DNA fragment that contains the ligated cohesive end (cos) site of bacteriophage .lambda., and a small size. A number of technical problems have prevented the wide spread use of this cloning technique. These problems, namely vector to vector ligation, "scrambling", and difficulties in screening large numbers of bacterial colonies can be dealt with using some recent advances by Meyerowitz et al. (see Gene 11: 271, 1980) and Grosveld et al. (see Gene 13: 220, 1981) Overall, the use of cosmids is most useful for certain specialized purposes such as isolation of large genes or for so called chromosome walking experiments.
The primary advantage of using bacteriophage M13 as a cloning vector is that the phage particles released from the cell contain single stranded DNA and therefore can be sequenced by the Sanger dideoxy-sequencing method (see Sanger et al. Proc. Nat. Acad. Sci., 74: 5463, 1977). However the relative instability of DNA inserts larger than about one kilobase effectively eliminates the usefulness of single stranded bacteriophages like M13 for most cloning purposes.
Transposons are discrete mobile DNA segments that are common constituents of plasmid, virus, and bacterial chromosomes. These elements are detected by their ability to transpose self encoded phenotypic traits from one replicon to another, or to transpose to a known gene and inactivate it. There are two types of transposons and they range in size from about 750 to greater than 50,000 nucleotide base pairs. One type known as the small insertion sequence or IS element are usually detected and were first discovered in the late 1960's as unusual insertion mutations. They do not encode any known phenotypic traits. The other type are relatively large units that do encode phenotypic traits such as antibiotic resistance. They were discovered in the mid 1970's. (See Plasmids and Transposons Environmental Effects and Maintenance Mechanisms; Edited by C. Stuttard and K. Rozee; Academic Press, New York; Pages 165-205)
Tn916 is a 10 megadalton transposable DNA element encoding resistance to tetracycline (Tc). It was originally identified on the chromosome of Streptococcus faecalis strain DS16 and is described in detail in Franke and Clewell, J. Bacteriol., 145: 494, (1981). Tn916 is unique because in addition to its ability to transpose into various plasmids, it has been shown to have fertility properties (Franke and Clewell J. Bacteriol., 145: 494, 1981 and Gawron-Burke and Clewell, Nature, 300: 281, 1982). Tn916 also has the unique property of readily excising under non selective conditions in an E. coli host.